Project Summary/Abstract: Platelet transfusion is an important therapeutic intervention for the treatment and prevention of bleeding. Unfortunately, some patients develop platelet refractoriness (PR), rapidly clearing transfused donor platelets and preventing the desired therapeutic outcome. Immune-mediated platelet clearance, attributed to recipient antiplatelet alloantibodies, plays a significant role in PR, and numerous studies demonstrate alloantibody-induced platelet clearance following transfusion. In the absence of antiplatelet antibodies, nonimmune causes of clearance can sometimes explain platelet loss. Still, many patients experience PR in the absence of a clear mechanism, suggesting that other processes of antibody-independent clearance may occur. Recently, we have found that CD8 T cells possess the ability to cause platelet clearance in the complete absence of antiplatelet alloantibodies. This finding provides an explanation as to why immune- mediated PR may be unresponsive to common approaches typically effective at preventing antibody-mediated platelet loss, and generates additional therapeutic targets for overcoming PR in patients. This proposal details a five-year research and training plan with a scientific focus on the mechanism by which CD8 T cells mediate clearance of transfused platelets. The long-term objective of this work is to understand antibody-independent PR, thereby allowing for optimal transfusion support of thrombocytopenic patients. The specific aims proposed here use three complementary approaches to understand the interaction between recipient CD8 T cells and transfused donor platelets. Aim 1 defines the CD8 T cell population responsible for allogeneic platelet loss. Aim 2 interrogates specific receptor-ligand interactions between alloreactive CD8 T cells and platelets. Aim 3 evaluates modifications in platelet glycans resulting from alloreactive CD8 T cell interaction and localizes the site of platelet clearance. These aims will not only advance our understanding of PR and identify possible therapeutic targets, but will also afford the additional training necessary for future studies as an independent blood scientist. A comprehensive mentoring team has been assembled for the completion of both the scientific and career development goals of this application: primary mentor Dr. Sean Stowell, MD, PhD, an expert in transfusion medicine and glycobiology research; co-mentor Dr. Rafi Ahmed, PhD, an expert in immunologic memory; co- mentor Mandy Ford, PhD, an expert in alloreactive T cell responses; and co-mentor Renhao Li, PhD, an expert in platelet biology. Through completion of the experiments and coursework outlined in this application, the immediate career development goals to gain basic and advanced training in platelet and CD8 T cell biology, as well exposure to bioinformatics, bioengineering, and glycobiology, will be attained. In addition, this K99 award will provide the preliminary data for successful R00 and, eventually, R01-level funding, launching an independent research career and thereby fulfilling long-term career goals to become a transfusion medicine specialist with an independently-funded basic science laboratory studying the intersection of hemostasis and adaptive immunity.